1. Field of the Invention
This invention relates to transfer and immobilization of nucleic acids, proteins, bacteria and viruses from electrophoresis gels or culture media to a porous membrane (film) for the purpose of probing the immobilized species for specific nucleic acids or proteins.
2. Description of the Prior Art
In a variety of procedures in analytical biochemistry it is necessary for the investigator to transfer a substance to be analyzed from a gel or culture medium to an immobilizing membrane for subsequent analysis.
In 1975, a method of transferring nucleic acids from electrophoresis gels to an adsorptive membrane was devised (Southern, E. M., J. Molecular Biology, 98, 503, (1975), and this general procedure is used widely in molecular biology laboratories. The Southern method involves placing filter paper on a glass plate elevated over a tray of buffer solution, with the ends of the paper extending into the buffer to form wicks; placing the gel containing the sample to be transferred on top of the filter paper; placing an adsorptive microporous membrane made of nitrocellulose on top of the gel; and placing paper towels on top of the membrane to blot the buffer and drive the sample into the membrane by capillary action. This is referred to as transfer by blotting.
In 1980, Bittner et al. (Anal. Biochem. 102, 459, (1980) described a modification in which an electrophoresis gel containing a sample to be transferred and an adsorptive nitrocellulose membrane are sandwiched between two sheets of filter paper, placed in an electrode cassette, immersed in a buffer solution, and subjected to an electrical potential. This is referred to as electrophoresis transfer or electroblotting.
Adsorptive microporous membranes have also been used to lift bacteria and viruses from growth media for subsequent manipulation and analysis of bacterial and viral nucleic acids and proteins.
Several materials have been used or suggested for use as transfer membranes, including nitrocellulose, nitrobenzyloxymethyl cellulose, aminobenzyloxymethyl cellulose, aminophenylthioether cellulose, diethylaminoethyl cellulose, and polyvinylidene fluoride.
Membranes of the cellulose-based materials are brittle and weak. They must be handled carefully in the transfer process. They cannot tolerate much of the manipulation involved in subsequent analytical work, such as hybridization of nucleic acids with radioactive probes, probe removal and rehybridization, immunological screening of proteins, lysis of cells, and autoradiography.
Microporous nylon films are used for filtration in many laboratories and industrial applications. Microporous nylon films have also been suggested (Hiratsuka et al. U.S. Pat. No. 4,128,470) for use as electrophoresis media in place of polyacrylamide gels in isoelectric focusing. Semi-permeable nylon membranes have been suggested for use in electroflocculation of suspended clay particles (Kunkle U.S. Pat. No. 4,048,038). Impermeable nylon films have been suggested as base sheets for microporous cellulose acetate or acetate/nitrate membrane electrophoresis media (Dwyer et al. U.S. Pat. Nos. 3,553,067, 3,594,263 and 3,759,773).